Abstract

Armed with the complete sequence of the human genome and an ever-increasing array of biological techniques, researchers continue to learn more about the genetic basis of diseases. For two decades, scientists and physicians have been developing therapeutic strategies for treating many diseases at the genetic level, creating the field of "gene therapy." For those diseases caused by loss-of-function mutations in a specific gene, delivery of a wild-type copy of that gene to affected cells can reduce or eliminate the disease phenotype. Viruses, having evolved to be extremely effective at delivering nucleic acids (i.e., their own genes for viral production) to cells, have been modified to include therapeutic genes of interest. While such viral gene therapy vectors are the most efficient vectors developed, concerns about their safety and immunogenicity have prompted many to investigate non-viral vector alternatives. Cationic polymers and lipids have emerged as leading non-viral vector materials. Our laboratory has developed a class of cyclodextrin-containing polycations (CDPs) that condense DNA into complexes that can be endocytosed by cells, achieve expression of their genetic payload in those cells, and may be modified to target particular cell types within an animal.

In the past five years, scientists have discovered a new mechanism for the reduction of gene expression in mammalian cells via sequence-specific cleavage of a particular messenger RNA (mRNA); this phenomenon is known as RNA interference (RNAi). Since RNAi is triggered by nucleic acids (small interfering RNA (siRNA) duplexes), I hypothesized that CDPs may be suitable vectors for the delivery of siRNA. In my thesis work, the safety of synthetic siRNA duplexes is examined both in cultured cells and in vivo. Using a number of different siRNA sequences, two different strains of mice, and three different methods of administration, I fail to observe any cytokine (IL-12 or IFN-a) responses, morphological changes, or alterations in complete blood counts (CBCs) or liver enzyme levels.

The ability of CDP to serve as a delivery vehicle for siRNA is also explored. I demonstrate that CDP/siRNA complexes can be formed that are small enough to be endocytosed, can be modified to ensure stability in physiological fluid, and protect the siRNA payload from serum nuclease degradation. Finally, down-regulation of specific target genes, including genes implicated in disease, is shown in vitro and in mice. An endogenous reporter gene (luciferase) in the livers of transgenic mice is down-regulated by galactosylated CDP/siRNA formulations that target hepatocytes. The level of a chimeric oncogene, EWS-Fli1, is reduced by polyplex formulations in cultured Ewing’s sarcoma cells and by transferrin-targeted formulations in tumor-bearing mice; this in vivo down-regulation corresponds to an inhibition of tumor growth. These results suggest that CDP-containing siRNA formulations have the potential for development into therapeutics.